The overall goal of this study is to develop vaccines that will protect male and female macaques from genital SIV transmission. Protection studies in macaques against intravenous challenges with SIV have been successful, but HIV vaccines for humans must also protect against a heterosexual route of infection. In our previous NCVDG studies, female macaques were protected against vaginal challenges. Protection was achieved when animals were immunized with formalin-treated SIV in biodegradable microspheres only if primed intramuscularly and boosted by oral (OR) or intratracheal (IT) routes. OR immunization alone did not protect. The data suggest that microspheres may serve as a basic vaccine strategy for protection against HIV vaginal transmission. This renewal application develops microsphere-based vaccines and other strategies for protection against heterosexual transmission, using the following specific aims: Aim 1. To identify the best of three immunization strategies for induction of vaginal antibody and to determine which strategy is best for protection against vaginal SIV challenge. The three strategies are intramuscular (IM) immunization only, IM priming plus OR boosting and IM priming plus IT boosting. Aim 2. To test recombinant SIV formulations consisting of SIV expressed proteins in microspheres for induction of vaginal IgG and IgA levels that are comparable to our previous results with whole SIV-microsphere immunizations. We will also determine if these SIV-recombinant- microsphere immunized animals are protected against vaginal challenge. Aim 3. To produce concentrated vaginal SIV antibody for testing its functions in neutralization and other immunological assays. Aim 4. To compare the thigh and arm muscles as intramuscular immunization sites for priming the induction of SIV vaginal immunity. Aim 5. To conduct immunogenicity studies in macaques using HIV recombinant antigens for inducing vaginal and systemic immunity. Aim 6. To develop a reliable male genital SIV challenge system.